Hello. Would somebody help me please? I'm desperate.
I am trying to express a human protein (enzyme) in human B-cell line
transformed with Epstein-Barr virus. I am using a vector pREP10 from
Stratagene. This vector carries oriP from EBV for its replication in the
cell and hygromycin phosphotransferase gene for selection of the
transformants. A cDNA encodong the enzyme of interest was ligated to pREP10
at downstream of RSV promoter, and the construct was introduced into the
cell by electroporation. After 3-day culture in the medium not containing
hygromycin B, the selection was begun. The problem is that all cells die
within a 2-week period of selection. The detailed conditions of
electroporation/selection are as follows:
1x107 (ten million) cells were suspended in 0.8 ml of PBS and 50 microgram
of the plasmid were added. The electroporatiion chamber was kept on ice for
10 min and electroporated with a capacitor discharge of 250 V and 960
microF. The chamber was incubated on ice for 10 min and cells were
transfered to a culture bottle containing 9.2 ml of RPMI 1640 medium
supplemented with 15% fetal calf serum. After culturing for 3 days,
selection for hygromycin B-resistant cells was begun with hygromycin B at
150 microgram/ml.
I would greatly appreciate hearing your suggestions/comments.